K562 is an erythroleukemic cell line used as a model for the study of the control of human globin gene expression. These cells do not support transcription of the beta-globin gene (human adult pattern of expression) but do express transcripts of epsilon- and gamma- globin genes (human embryonic and fetal pattern) at very high levels when exposed to a number of inducing agents. Results from this and other laboratories suggest that the control of this pattern of expression is mediated by the presence and/or absence of trans-acting factors which exert their action on sequences corresponding to the promoters of these genes. Sequence specific DNA binding proteins acting on cis-regulatory elements have been hypothesized to be key elements in eukaryotic gene transcription, and even though considerable progress has been made in their isolation, DNA binding proteins with affinity for the human globin gene promoters have not yet been identified. We have defined several positive and negative regulatory regions 5' to the epsilon- globin gene promoter, and detected binding of proteins to these regions. The methodology used included DNase footprinting and the gel retardation assay. One of the negative regions defined binds to a factor present in high concentrations in non-erythroid cells. Using in vitro mutagenesis, inhibition of binding of a protein to this region causes a 10 fold activation of the epsilon-globin gene promoter in a CAT-assay. This technique has also allowed us to further define several positive and negative regulatory sequences 5' to the protein coding region of this gene.